This invention concerns compositions and methods for the detection of analytes which may be present in a wide range of possible concentrations through the use of two or more labeled binding partners. In a particular embodiment, two or more separately distinguishable labels are each joined to binding partners designed to bind two mutually exclusive target regions of the same analyte. In this preferred embodiment, the label joined to each said binding partner is present at a different specific activity. In other embodiments, the labels are present on the binding partners at the same specific activity, or are not separately distinguishable. The present invention provides for the extension of the dynamic range of any assay to be used for detection of any analyte, using any label type or combination of label types. The binding partners must be able to bind the analyte in a target region-specific manner; otherwise the binding partners may be of any type. The invention is useful in any application in which the detection of analyte is desired: these include, without limitation, nucleic acid hybridization assays, antibody binding assays, enzyme-substrate interactions, cytokine-receptor interactions, hapten-ligand interactions, and the like.
Assays for the detection and/or quantification of analytes exist in many different forms and formats. In many cases, the amount of the analyte sought to be detected in a sample is not great enough for direct detection or quantification. In such cases, a secondary molecule able to bind to or interact with the analyte must be used to indicate the presence or amount of analyte in a given sample. Unless the secondary molecule is directly detectable, the secondary molecule must be conjugated with a label which will be detected when the secondary molecule binds the analyte. For the purposes of the present application, such secondary molecules able to bind or interact with analytes will be referred to as xe2x80x9cbinding partnersxe2x80x9d or xe2x80x9cprobesxe2x80x9d. A non-exclusive list of analytes sought to be detected are antibodies, proteins, cell-surface receptors, cytokines, hormones, antigens, nucleic acids, metals, molecular complexes such as polymeric arrangements of proteins or other macromolecules, and the like. Likewise, binding partners for the detection of such analytes may include, without limitation: antibodies, proteins, antigens, haptens, nucleic acid probes, chelating agents, enzymes, enzyme substrates, and analogs of these.
One commonly used assay format is the enzyme-linked immuno-absorption assay (ELISA). In this assay format the analyte is contacted with a primary antibody able to bind at least one domain or xe2x80x9ctarget regionxe2x80x9d thereon. After the excess antibody is washed free of the resulting analyte: antibody complex, the primary antibody is contacted with an enzyme-labeled secondary antibody to which it will specifically bind. The sample is then given a chromogenic enzyme substrate, and incubated under conditions favoring enzyme-mediated reaction of the substrate. The resulting colored product and its intensity after a given reaction time are indications of the presence and amount, respectively, of the analyte originally present in the sample. Illustrative examples of enzymes used in such assay methods are xcex2-galactosidase, acid phosphatase, and alkaline phosphatase; a non-exhaustive list of enzyme substrates for use with such enzymes include x-gal (5-bromo-4-chloro-3-indolyl-xcex2-D-galacto-pyranoside) and p-nitrophenyl phosphate. Other such enzymes and substrates are well known by those of skill in the art. Variations of this assay method exist; for example, the primary antibody may be linked to an enzyme thus eliminating the secondary antibody step. Nevertheless, these assay methods feature common steps involving contacting of the analyte with a labeled binding partner and subsequent detection of the analyte-bound label as an indication of the presence or amount of analyte.
While ELISA utilizes an enzyme label which is indirectly detected, other methods exist for the direct detection of labeled binding partner. Thus, Campbell, et al., U.S. Pat. No. 4,496,958 describes chemiluminescent acridinium labeling compounds for use in labeling binding partners in multiple assay formats; this patent is incorporated by reference as part of the present application. Additionally, other labeling compounds such as radionuclides, fluorescent, bioluminescent, phosphorescent, luminescent, chemiluminescent, or electrochemiluminescent compounds, chromophores, and dyes are known in the art and are commonly used as labeling agents in a variety of assay formats, both direct and indirect, including immunoassay and nucleic acid hybridization assays.
In addition to distinctions between assays based on the type of analytes to be detected, the type of binding partner with which the analyte binds, and the type of label used, assays can also be classified according to whether the method involves the immobilization of the analyte, or the analyte: binding partner complex. In the most common assay format, known as a xe2x80x9cheterogeneousxe2x80x9d or biphasic assay system, a probe is allowed to bind its analytexe2x80x94usually under conditions of probe excess. Either the analyte molecule or the probe molecule may be immobilized to a solid support, thus causing the resulting probe: analyte complex to become immobilizedxe2x80x94alternatively, and preferably, the complex may be immobilized following its formation in the liquid phase. After probe: analyte complexes have been immobilized, the excess uncomplexed probe molecules are washed away. If the probe molecules are directly labeled, the label may now be detected as an indication of the presence of analyte. In a variation of this format, probe: analyte complexes may also be separated from free probe, by means such as gel filtration chromatography, electrophoresis, electrofocusing, and other separation methods based on size or charge of the probe: analyte complex.
Alternatively, and more rarely, an assay may be designed to take place wholly in a single phase without a step resulting in the physical separation of probe: analyte complex from free probe. Such assay methods are termed xe2x80x9chomogeneousxe2x80x9d assays. In such methods, usually either the analyte: probe complex, or the free probe is altered after formation of the complex to permit the separate detection of analyte in the presence of the free probe. One such way of differentiating free probe from probe-bound analyte involves alteration or selective inactivation of the label joined to the probe rather than the free probe molecule itself. Arnold, et al., U.S. Pat. No. 5,283,174, describes homogeneous methods employing a oligonucleotide probe joined to a label which is capable of selective inactivation or alteration based on whether the labeled probe is bound to its target or not. These methods may be used in a single tube without the need for washing or decanting. This patent enjoys common ownership with the present application, and is incorporated by reference herein.
Assay methods exist which may utilize aspects of both homogeneous and heterogeneous assays. These methods may, for example, employ a single phase selective alteration of the probe or the probe: analyte complex followed by a physical separation step to further decrease the level of background in the assay. Such a xe2x80x9chybridxe2x80x9d assay format is described as one aspect of the multiple analyte assay described in Nelson et al., U.S. Pat. No. 5,658,737 which is incorporated by reference herein.
Regardless of the assay format used, a number of factors exist in all assays which can limit their sensitivity and the range of possible analyte concentrations that can be accurately detected or measured. One such factor is the level of background present in the assay. xe2x80x9cBackgroundxe2x80x9d is a term used to describe probe or label in the assay which is not bound specifically to analyte and which may mask positive results at low analyte levels. Thus, for example, in a heterogeneous assay, background may be provided by a small amount of probe which is not removed during the physical separation of probe: analyte complex from free probe. If the probe is labeled, this small amount of probe will be detected as a residual level of detectable signal. In a homogeneous assay, background may be provided by the inability to totally alter all free probe or, probe-linked label molecules. In either case, a small level of signal, commonly between 0.001% and 10% of the total signal, more commonly between about 0.01% and 1%, is present as a xe2x80x9cbaselinexe2x80x9d below which results cannot be relied on for accuracy. Thus, the level of background inherent in a particular assay format limits the lowest amount of analyte which can be detected and/or measured.
The phenomenon of residual background in an assay plays a part in limiting the xe2x80x9cdynamic rangexe2x80x9d of that particular assay. By xe2x80x9cdynamic rangexe2x80x9d is meant a linear or predictably accurate correspondence between the level of analyte present in the sample to be assayed and the amount of signal obtained from the label used to indicate the analyte""s presence. It is readily apparent to those of skill in the art that the dynamic range of an assay cannot extend below the level of background contributed by the detection of non-specific label, thus the higher the background, the more the dynamic range is limited. Moreover, if high amounts of analyte are to be detected, correspondingly high amounts of probe have to be used, which leads to higher backgrounds.
Other factors may contribute to limitations on the extent of a particular assay""s dynamic range. A major additional factor is often the maximum amount of signal able to be read or reported by the label detection device or instrument to be used in the assay. Thus, if a given instrument can only accurately read up to, for example, one million counts per second and the sample yields 2 million counts per second, the extra one million counts are not being reported by the instrument, and the upper extent of the assay dynamic range is half of what is necessary to accurately quantify the sample.
Additionally, instruments used to detect the labeled probe: analyte complexes may have inherent electronic xe2x80x9cnoisexe2x80x9d, which is also commonly termed xe2x80x9cbackgroundxe2x80x9d, and which can also contribute to limitations on the accuracy of the detection of the analyte. While improvements to, and optimization of an instrument""s electronic signal-to-noise ratio are possible, the noise obtained in a specific instrument combines with the inherent assay background described above to further limit the ability to detect or quantify analytes across a wide range of possible concentrations. In order to overcome these limitations, it is currently necessary for workers to obtain multiple samples from a single source, or to make serial dilutions of a sample to test for the presence of an unknown amount of analyte.
There is, therefore, currently a need in the art for methods and compositions for detecting and/or quantifying analytes of every kind in a single tube without the need for sample dilutions or duplicate samples. Preferably, such methods and compositions would involve a single addition of detection probes from which possible analyte concentrations differing by orders of magnitude can be detected. Such methods should also generally be independent of the analyte type, probe type, label type, and instrument to be used for the detection of analyte.
The present invention is directed to methods and compositions permitting the detection of at least one analyte over a broader total concentration range than is otherwise commonly possible. In its most basic form, the invention utilizes two or more labeled probes, each probe targeted to the same analyte in a different target region. Each labeled probe is used to detect analyte within a different specified concentration range, and is present in the assay in an amount which corresponds in a defined manner to the maximum molar amount of analyte within that range. Thus, the labeled probe molecules are present in the assay at different concentrations, which correspond to the different analyte concentration ranges sought to be detected. In an embodiment of the invention, the signal produced by each of the labels can be indistinguishable; however, in a preferred embodiment the signal produced by each label is independently distinguishable. Furthermore, each probe may be labeled at the same, or preferably at different specific activities.
Thus, in a first aspect, the invention concerns a method of detecting analytes and extending the range of concentrations at which said analytes can be detected, by using two or more probe molecules labeled with a detectable label. Each probe molecule is directed to a different target region of the same analyte.
Preferably, the labels to which the probe molecules are joined are separately detectable. By xe2x80x9cseparately detectablexe2x80x9d is meant that the labels can be present in the same reaction vessel and each distinguished in the presence of the other. Such labels may be of the same general type, such as radionuclides (for example, 32P and 125I; the first being detectable by emission of xcex2 particles and the other by emission of xcex3 rays), and different chemiluminescent or fluorescent labels. Alternatively, one probe may be labeled with a particular type of label and another probe may be labeled with a different type of label, such as the first with a radionuclide and the second with a fluorescent label. In less preferred embodiments the labels used to xe2x80x9ctagxe2x80x9d the probes need not be separately detectable so long as they are present on each probe at different specific activities.
By xe2x80x9clabeledxe2x80x9d is meant that a probe is joined to a labeling compound. The label can be joined either directly or indirectly. An example of indirect labeling is through the use of a bridging molecule, such as a secondary antibody or a bridging oligonucleotide, which is itself either directly or indirectly labeled. Direct labeling can occur through covalent bond formation or through non-covalent interactions such as hydrogen bonding, hydrophobic and ionic interactions, or through the formation of chelates or coordination complexes.
When reference is made to the concentration or amount of a particular probe corresponding to an amount of analyte which the probe is designed to detect, it will be understood that by xe2x80x9ccorresponding toxe2x80x9d is meant that the concentrations or amounts of the probe and analyte are related in a predictable and reproducable way; for example, by a given ratio, under a given set of assay conditions.
By xe2x80x9coligonucleotidexe2x80x9d is meant a multimeric compound comprised of nucleosides or nucleoside analogs which have nitrogenous bases, or base analogs, able to specifically bind a nucleic acid analyte to form a stable probe:analyte complex. The nucleosides may be linked together by phosphodiester bonds to form a polynucleotide, or may be linked in any other manner that permits the formation of a target-specific complex with a target nucleic acid. Other internucleoside linkages may include phosphorothioate linkages, methylphosphonate linkages, and peptide bonds. Peptide nucleic acids are defined herein as oligonucleotides. Sugar moieties may be substituted with groups affecting stability of the hybridization reaction (but not extinguishing formation of a target:probe complex), as, for example, with 2xe2x80x2 methoxy substitutions and 2xe2x80x2 halide substitutions such as 2xe2x80x2-F. Similarly, while the bases may consist of the xe2x80x9ctraditionalxe2x80x9d bases adenine, guanine, cytosine, uracil and thymine, analogs of these bases are well known in the art, (see, e.g., The Biochemistry of the Nucleic Acids 5-36 (Adams et al., ed. 11th ed. 1992), which is hereby incorporated by reference herein), and are contemplated to be within the scope of this term.
By xe2x80x9cspecific activityxe2x80x9d is meant units of detectable signal obtained from a label per unit measurement of probe. The units of detectable signal may be expressed in counts per minute (cpm), light absorbance units at a given wavelength, relative light units (rlu), units of enzymatic activity or any other unit of measuring the amount of label present. Likewise, the units of probe measurement may be expressed as units of mass, such as xcexcg, or as a measurement of the number of molecules of probe, such as xcexcmoles.
By the terms xe2x80x9cbinding partnersxe2x80x9d or xe2x80x9cprobesxe2x80x9d is meant a molecule able to bind to or interact with the analyte. A probe or binding partner is used to indicate the presence or amount of analyte in a given sample. Unless the probe or binding partner is directly detectable, the probe is directly or indirectly conjugated with a label. A non-exclusive list of analytes which may be sought to be detected are antibodies, proteins, enzymes, lipids, carbohydrates, cell-surface receptors, cytokines, hormones, antigens, nucleic acids, metals, molecular complexes such as polymeric arrangements of proteins or other macromolecules, and the like. Likewise, binding partners for the detection of such analytes may include antibodies, antigens, haptens, nucleic acid probes, chelating agents, enzymes, enzyme substrates, proteins and analogs of these.
By xe2x80x9ctarget regionxe2x80x9d or xe2x80x9cregionxe2x80x9d is meant any portion of an analyte, continuous or discontinuous, which binds to a given probe (or binding partner) or class of probes. For example, without limitation, when the analyte is a nucleic acid molecule, a target region may comprise a nucleotide base sequence which will specifically bind a probe. The target region may also comprise a particular secondary or tertiary structure of the target analyte to which a probe is directed. If the analyte is a protein or peptide, the target region may be an amino acid sequence or a conformational domain within the protein or peptide that can bind to a probe. Preferably, each target region does not overlap any other target region of the same analyte, so that simultaneous application of each probe to the analyte will not produce competition between probes.
It is also, therefore, an object of the invention to provide methods for the detection of a wide range of target analyte concentrations. Typically, these methods can increase the ability to accurately detect the analyte by at least two orders of magnitude; however through the use of additional probes and separately distinguishable labels, the Applicant contemplates that the dynamic range of an assay may be extended by 3-6 orders of magnitude or more. Such methods allow the detection of analyte in a sample without the need to perform sample dilutions or to test replicate samples under different conditions. This is possible since according to the present invention, the dynamic response of the assay to expected analyte levels is designed a priori to cover desired ranges of analyte amounts. Thus, preferred aspects of the invention provide a method for the analysis of a sample in a single tube.
It is further an object of the present invention to provide compositions for the detection or quantification of an analyte that can be used by a person with a minimum of specialized training in medical, clinical, or scientific methodology. By permitting the testing of an analyte which may be present in a sample at a concentration anywhere within a wide range of potential concentrations, the invention provides compositions which can permit the automation of many aspects of diagnostic assays while minimizing sample handling and the attendant heightened risk of error. Thus, the present invention provides useful means for minimizing the level of specialized training necessary for laboratory personnel to conduct assays, while permitting such assays to be easily automated with reduced variability of results.
A further object of the present invention is to provide a method for detecting or measuring one or more analyte suspected of being present in a sample comprising the steps:
a) contacting
i) said sample, and
ii) a probe reagent comprising two or more probes, each said two or more probes joined to a label and able to selectively bind to a separate target region of said analyte, wherein each said labeled probe is designed to detect said analyte over a different range of analyte concentrations and wherein the amount of each said probe present in said probe reagent corresponds to the amount of said analyte sought to be detected by that probe,
under conditions favoring the binding of said probe molecules with said analyte, if present, and
b) detecting the presence of at least one said label as an indication of the presence or amount of said analyte in said sample,
wherein the range of possible analyte amounts in said sample able to be detected or measured by said probe reagent is greater than the range of analyte amounts in said sample able to be detected or measured by any one said labeled probe, and wherein both said contacting and detecting steps are capable of being performed in the same vessel. In this embodiment, the invention may comprise the use of separately distinguishable labels. Additionally, and independently, the invention may comprise the use of two or more labeled probes having different specific activities.
Probes are present in the probe reagent in different amounts. The amount of each probe corresponds to and defines the upper limit of the range of analyte concentrations which that labeled probe is designed to detect. This is a greatly preferred feature of the invention, since the amount of each labeled probe in the assay can define the amount of background present. Additionally, when the probes differ in their specific activities, the probe having the highest specific activity is generally present in the lowest amount, while the probe having the lowest specific activity is generally present in the highest amount. In this way, background levels from label associated with unbound probe are minimized.
It is yet further an object of the invention to provide compositions and kits for detecting analytes which may be present in a sample within a wide range of possible concentrations. Such compositions may be drawn to a probe reagent comprising two or more probes, each probe being joined to a label and designed to bind to a separate target region of said analyte, wherein each said labeled probe is designed to detect said analyte over a different range of possible analyte concentrations and wherein the concentration of each said probe present in the reagent corresponds to the concentration the analyte sought to be detected.
In each of these embodiments, the analyte may comprise any molecule or complex capable of binding two or more probes. Likewise, a probe may comprise any molecule capable of specifically binding to a target region of the analyte. Each of the probe and the analyte may comprise compounds including an antigen, an antibody, a hormone, a protein, a cytokine, an oligonucleotide, a cellular receptor, a nucleic acid, and a peptide nucleic acid.